NAD:arginine ADP-ribosyltransferases catalyze the ADP-ribosylation of arginine residue(s) in proteins. Coding region nucleic acid and deduced amino acid sequences of a human skeletal muscle ADP-ribosyltransferase cDNA were respectively, 80.8% and 81.3% identical to those of the rabbit skeletal muscle transferase. A human transferase-specific cDNA probe detected a predominant mRNA of 1.2 kb (mouse and rat), 3.0 kb (rabbit), 3.8 kb (monkey) and 5.7 kb (human) on Northern analysis. Polyclonal anti-rabbit ADP-ribosyltransferase antibodies reacted with 36,000 Mr- proteins in partially purified transferase preparations from bovine, dog and rabbit heart muscle and a 40,000-Mr protein from human skeletal muscle. The human muscle ADP-ribosyltransferase cDNA, like the previously cloned rabbit muscle transferase, predicts predominantly hydrophobic amino- and carboxy-terminal amino acid sequences, characteristic of glycosylphosphatidylinositol (GPI)-anchored proteins. On immunoblots of partially purified rabbit and human skeletal muscle ADP-ribosyltransferases, anti-cross-reacting determinant antibodies detected at 36,000 Mr and 40,000 Mr, respectively, phosphatidylinositol- specific phospholipase C-sensitive GPI-anchored proteins. These data are consistent with the conclusion that GPI-anchored skeletal and cardiac muscle ADP-ribosyltransferases are conserved among mammalian species.